We have previously demonstrated cancer stem cell (CSC) subpopulations in head and neck metastatic malignant melanoma (HNmMM), and the expression of components of the renin-angiotensin system (RAS) by these CSCs. Cathepsins B, D and G are involved in carcinogenesis and constitute bypass loops of the RAS. This study investigated the expression and localization of cathepsins B, D and G, in relation to these CSCs. Immunohistochemical staining demonstrated expression of cathepsins B, D and G in HNmMM sections from all 20 patients. Western blotting confirmed the presence of cathepsins B and D proteins in all six HNmMM tissue samples and four HNmMM-derived primary cell lines. RT-qPCR showed transcript expression of cathepsins B, D and G in all six HNmMM tissue samples, and cathepsins B and D but not cathepsin G in all four HNmMM-derived primary cell lines. Enzymatic activity assays demonstrated cathepsins B and D were active in all six HNmMM tissue samples. Immunofluorescence staining performed on two of the HNmMM tissue samples demonstrated expression of cathepsins B and D by the CSCs, and cathepsin G by cells within the peritumoral stroma. Our novel findings suggest the possibility of targeting these CSCs by modulation of paracrine RAS signaling.
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